Small RNA Library Construction from Minute Biological Samples

Abstract

Increasingly, the discovery and characterization of small regulatory RNAs from a variety of organisms have all required deep-sequencing methodologies. However, the crux to successful deep-sequencing analysis depends upon optimal construction of a cDNA library compatible for the high-throughput sequencing platform. Challenges to small RNA library constructions arise when dealing with minute tissue samples because certain structural RNA fragments can dominate and mask the desired characterization of regulatory small RNAs like microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs). Here, we describe methods that improve the chances of constructing a successful library from small RNAs isolated from minute tissues such as enriched follicle cells from the Drosophila ovarium. Because the ribosomal RNA (rRNA) fragments are frequently the major contaminants in small RNA preparations from minute amounts of tissue, we demonstrate the utility of antisense oligonucleotide depletion and an acryloylaminophenylboronic acid (APB) polyacrylamide gel system for separating the abundant 2S rRNA in Drosophila from endo-siRNAs and piRNAs. Finally, our methodology generates libraries amenable to multiplex sequencing on the Illumina Hi-Seq platform.

Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution

“…Next, we performed RNA-seq on ovarian follicle cells 31 isolated from D. melanogaster and D. yakuba adult females (Supplementary Tables 3 and 4). Gene expression levels were very similar in both species (PCC = 0.89), consistent with the high conservation of gene expression reported for fly embryos 32 and mammals 11 .…”

“…Samples highly enriched in follicle cells from D. melanogaster (OregonR) and D. yakuba (WT Liberia; obtained from DSSC) were prepared as described previously 31 . Fly strains were maintained under standard fly culture procedures, but D. yakuba were given a moist substrate in bottles to facilitate pupation.…”

Transposable element dynamics and PIWI regulation impacts lncRNA and gene expression diversity in Drosophila ovarian cell cultures

“…RNA from the NUN fraction was depleted from ribosomal RNA using a biotinylated oligo set (Pennington et al 2013). RNA-seq libraries were obtained from four independent PIWI knockdown experiments using four different library construction protocols: (1) ScriptSeq V1; (2) ScriptSeq V2 (Epicenter; performed according to manufacturer's instructions); (3) random primer-based library construction protocol (Pennington et al 2013); and (4) mRNA fragmentation followed by the small RNA library construction protocol (sRNA-seq) (Matts et al 2014). Sequencing was performed on an Illumina HiSeq 2000, and 50-bp-long reads were processed and split according to their index primer barcodes.…”

Improved Boronate Affinity Electrophoresis by Optimization of the Running Buffer for a Single-step Separation of piRNA from Mouse Testis Total RNA

“…14 Alternatively, Lau et al recently exploited the boronate affinity electrophoresis for discriminating piRNAs from other small RNAs in follicle cells from Drosophila ovarium. 15 The principle is based on specific recognition of boronic acid for the cis-diol group of RNA ribose in gel electrophoresis, as used for the analysis of a 3′-terminal modification of longer RNAs. 16,17 Thus, RNA species having free 3′-terminal ribose can be captured by boronic acids in the gels, whereas piRNAs with 2′-O-methylation can not.…”

“…18 Isolated piRNAs can be readily subject to a downstream analysis, such as deep-sequencing. 15 Therefore, this approach benefits from simple and easy operation for piRNA separation compared to the above-mentioned chemical treatment. In this work, we further improved the boronate affinity electrophoresis for piRNA separation based on an examination of the running buffer, demonstrating a single-step piRNA separation from non-purified total RNA.…”